Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control.

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.

Water potential governs the effector specificity of the transcriptional regulator XylR of Pseudomonas putida.

The biodegradative capacity of bacteria in their natural habitats is affected by water availability. In this work, we have examined the activity and effector specificity of the transcriptional regulator XylR of the TOL plasmid pWW0 of Pseudomonas putida mt-2 for biodegradation of m-xylene when external water potential was manipulated with polyethylene glycol PEG8000. By using non-disruptive luxCDEAB reporter technology, we noticed that the promoter activated by XylR (Pu) restricted its activity and the regulator became more effector-specific towards head TOL substrates when cells were grown under water subsaturation. Such a tight specificity brought about by water limitation was relaxed when intracellular osmotic stress was counteracted by the external addition of the compatible solute glycine betaine. With these facts in hand, XylR variants isolated earlier as effector-specificity responders to the non-substrate 1,2,4-trichlorobenzene under high matric stress were re-examined and found to be unaffected by water potential in vivo. All these phenomena could be ultimately explained as the result of water potential-dependent conformational changes in the A domain of XylR and its effector-binding pocket, as suggested by AlphaFold prediction of protein structures. The consequences of this scenario for the evolution of specificities in regulators and the emergence of catabolic pathways are discussed.

[Pharmacotherapy for children and adolescents with sleep disorders: an overview]. / Psychopharmakotherapie bei Ein- und Durchschlafstörungen im Kindes- und Jugendalter: Eine Übersicht.

Pharmacotherapy for children and adolescents with sleep disorders: an overview Abstract. Sleep difficulties are a relevant symptom in childhood and adolescence with a prevalence of approximately 20 %. Because of the reduced psychosocial functioning and the burden of suffering associated with insomnia, effective treatment strategies are needed. If interventions such as the implementation of an adequate sleep hygiene and other non-drug treatment approaches are not sufficient, pharmacotherapeutic treatment is often considered. The present work provides a literature overview on the use of different substance classes in hypnotic indications. The use of melatonin in children and adolescents with autism spectrum disorder has been well studied and is associated with a positive effect on sleep in this patient group. To date, there is little evidence regarding the efficacy and tolerability of other medication in primary insomnia or sleep disorders in the context of other psychiatric disorders in minors. For this reason, non-drug treatment strategies are preferred and pharmacotherapy is only to be considered secondarily after critical examination.

Prevalence Rate and Course of Symptoms of Disruptive Mood Dysregulation Disorder (DMDD).

Objective: According to DSM-5, Disruptive Mood Dysregulation Disorder (DMDD) is characterized by chronic temper outbursts and irritable moods. So far, little is known about its prevalence rate, course and influence on individual well-being. We assessed the prevalence rates of DMDD symptoms during adulthood and primary school age - the latter retrospectively - and studied their relationship with psychiatric disorders and socioeconomic variables. Methods: A total of 2,413 subjects, aged 18-94 years, participated in this population-based, representative study based on self-reports. Results: 12 (0.50 %) subjects reported elevated DMDD symptoms during adulthood, and 19 (0.79 %) reported elevated DMDD symptoms during primary school age. DMDD symptoms were associated with higher rates of depression and anxiety symptoms. Those reporting elevated DMDD symptoms during adulthood were more often single or divorced, and those reporting elevated DMDD symptoms during primary school age were more often childless and unemployed during adulthood compared to subjects without DMDD symptoms. Conclusions: DMDD symptoms seem to show a chronic course and go hand in hand with elevated psychiatric symptoms and impaired socioeconomic and demographic status.

Mild Hypothermia in a Child with Low-Dose Risperidone.

Risperidone is a widely used, second-generation antipsychotic approved for treating schizophrenia as well as for treating aggression in children and adolescents with mental retardation. The substance has a well-established risk profile including alterations of body temperature. Apart from hyperthermia with and without full-blown malignant neuroleptic syndrome, low body temperatures (hypothermia) have also been reported anecdotally, usually appearing in the context of comedication. Here, we report a case of hypothermia associated with a low-dose risperidone monotherapy in a child.

[Body dysmorphic disorder]. / Körperdysmorphe störung.

Body dysmorphic disorder (BDD) is a relatively common disorder with a point prevalence of 0.7-2.4 %. BDD is characterized by the patient's excessive concern with an imagined or slight defect in physical appearance. BDD usually begins in adolescence. Comorbidity rates and also suicidality rates are high. The course of BDD tends to be chronic. According to the present state of knowledge, cognitive-behavioral therapy and pharmacotherapy with selective serotonin reuptake inhibitors are valuable options in the therapy of BDD. The case report describes a recent case of BDD with typical clinical and therapy-related characteristics. The aim of this work is to strengthen the awareness of BDD in clinical practice of child and adolescent psychiatry, facilitating an adequate diagnosis and treatment of the affected individuals.

Reproducibility of CT-based opportunistic vertebral volumetric bone mineral density measurements from an automated segmentation framework.

BACKGROUND: To investigate the reproducibility of automated volumetric bone mineral density (vBMD) measurements from routine thoracoabdominal computed tomography (CT) assessed with segmentations by a convolutional neural network and automated correction of contrast phases, on diverse scanners, with scanner-specific asynchronous or scanner-agnostic calibrations. METHODS: We obtained 679 observations from 278 CT scans in 121 patients (77 males, 63.6%) studied from 04/2019 to 06/2020. Observations consisted of two vBMD measurements from Δdifferent reconstruction kernels (n = 169), Δcontrast phases (n = 133), scan Δsessions (n = 123), Δscanners (n = 63), or Δall of the aforementioned (n = 20), and observations lacking scanner-specific calibration (n = 171). Precision was assessed using root-mean-square error (RMSE) and root-mean-square coefficient of variation (RMSCV). Cross-measurement agreement was assessed using Bland-Altman plots; outliers within 95% confidence interval of the limits of agreement were reviewed. RESULTS: Repeated measurements from Δdifferent reconstruction kernels were highly precise (RMSE 3.0 mg/cm3; RMSCV 1.3%), even for consecutive scans with different Δcontrast phases (RMSCV 2.9%). Measurements from different Δscan sessions or Δscanners showed decreased precision (RMSCV 4.7% and 4.9%, respectively). Plot-review identified 12 outliers from different scan Δsessions, with signs of hydropic decompensation. Observations with Δall differences showed decreased precision compared to those lacking scanner-specific calibration (RMSCV 5.9 and 3.7, respectively). CONCLUSION: Automatic vBMD assessment from routine CT is precise across varying setups, when calibrated appropriately. Low precision was found in patients with signs of new or worsening hydropic decompensation, what should be considered an exclusion criterion for both opportunistic and dedicated quantitative CT. RELEVANCE STATEMENT: Automated CT-based vBMD measurements are precise in various scenarios, including cross-session and cross-scanner settings, and may therefore facilitate opportunistic screening for osteoporosis and surveillance of BMD in patients undergoing routine clinical CT scans. KEY POINTS: Artificial intelligence-based tools facilitate BMD measurements in routine clinical CT datasets. Automated BMD measurements are highly reproducible in various settings. Reliable, automated opportunistic osteoporosis diagnostics allow for large-scale application.

Pseudomonas putida as saviour for troubled Synechococcus elongatus in a synthetic co-culture - interaction studies based on a multi-OMICs approach.

In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of the cyanobacterium S. elongatus cscB, which supplies sucrose to the heterotrophic P. putida cscRABY, is investigated to identify potential interactions. Initial experiments reveal a remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa, the presence of the cyanobacterium has a neutral effect on P. putida cscRABY, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next, a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system.

Cra regulates the cross-talk between the two branches of the phosphoenolpyruvate : phosphotransferase system of Pseudomonas putida.

The gene that encodes the catabolite repressor/activator, Cra (FruR), of Pseudomonas putida is divergent from the fruBKA operon for the uptake of fructose via the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS(Fru)). The expression of the fru cluster has been studied in cells growing on substrates that change the intracellular concentrations of fructose-1-P (F1P), the principal metabolic intermediate that counteracts the DNA-binding ability of Cra on an upstream operator. While the levels of the regulator were not affected by any of the growth conditions tested, the transcription of fruB was stimulated by fructose but not by the gluconeogenic substrate, succinate. The analysis of the P(fruB) promoter activity in a strain lacking the Cra protein and the determination of key metabolites revealed that this regulator represses the expression of PTS(Fru) in a fashion that is dependent on the endogenous concentrations of F1P. Because FruB (i.e. the EI-HPr-EIIA(Fru) polyprotein) can deliver a high-energy phosphate to the EIIA(Ntr) (PtsN) enzyme of the PTS(Ntr) branch, the cross-talk between the two phosphotransferase systems was examined under metabolic regimes that allowed for the high or low transcription of the fruBKA operon. While fructose caused cross-talk, succinate prevented it almost completely. Furthermore, PtsN phosphorylation by FruB occurred in a Δcra mutant regardless of growth conditions. These results traced the occurrence of the cross-talk to intracellular pools of Cra effectors, in particular F1P. The Cra/F1P duo seems to not only control the expression of the PTS(Fru) but also checks the activity of the PTS(Ntr) in vivo.

Streamlining of a synthetic co-culture towards an individually controllable one-pot process for polyhydroxyalkanoate production from light and CO2.

Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications. The application of such a consortium in a bioprocess, however, requires tight and individual controllability of the involved microbes. Here, we present the streamlining of a co-cultivation process consisting of Synechococcus elongatus cscB and Pseudomonas putida for the production of polyhydroxyalkanoates (PHA) from light and CO2. First, the process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually, and defined conditions could be set. The molC/molN ratio, a key value for PHA accumulation, was estimated from the experimental data, and the necessary feeding rates to obtain a specific ratio could be predicted. This information was then implemented in the co-cultivation process, following the concept of a DBTL-cycle. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg/L and a PHA production rate of 42.1 mg/(L•day).

The interplay of the EIIA(Ntr) component of the nitrogen-related phosphotransferase system (PTS(Ntr)) of Pseudomonas putida with pyruvate dehydrogenase.

BACKGROUND: Pseudomonas putida KT2440 is endowed with a variant of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS(Ntr)), which is not related to sugar transport but believed to rule the metabolic balance of carbon vs. nitrogen. The metabolic targets of such a system are largely unknown. METHODS: Dielectric breakdown of P. putida cells grown in rich medium revealed the presence of forms of the EIIA(Ntr) (PtsN) component of PTS(Ntr), which were strongly associated to other cytoplasmic proteins. To investigate such intracellular partners of EIIA(Ntr), a soluble protein extract of bacteria bearing an E epitope tagged version of PtsN was immunoprecipitated with a monoclonal anti-E antibody and the pulled-down proteins identified by mass spectrometry. RESULTS: The E1 subunit of the pyruvate dehydrogenase (PDH) complex, the product of the aceE gene, was identified as a major interaction partner of EIIA(Ntr). To examine the effect of EIIA(Ntr) on PDH, the enzyme activity was measured in extracts of isogenic ptsN(+)/ptsN(-)P. putida strains and the role of phosphorylation was determined. Expression of PtsN and AceE proteins fused to different fluorescent moieties and confocal laser microscopy indicated a significant co-localization of the two proteins in the bacterial cytoplasm. CONCLUSION: EIIA(Ntr) down-regulates PDH activity. Both genetic and biochemical evidence revealed that the non-phosphorylated form of PtsN is the protein species that inhibits PDH. GENERAL SIGNIFICANCE: EIIA(Ntr) takes part in the node of C metabolism that checks the flux of carbon from carbohydrates into the Krebs cycle by means of direct protein-protein interactions with AceE. This type of control might connect metabolism to many other cellular functions. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Targeting Transcriptional and Translational Hindrances in a Modular T7RNAP Expression System in Engineered Pseudomonas putida.

The T7 RNA polymerase is considered one of the most popular tools for heterologous gene expression in the gold standard biotechnological host Escherichia coli. However, the exploitation of this tool in other prospective hosts, such as the biotechnologically relevant bacterium Pseudomonas putida, is still very scarce. The majority of the existing T7-based systems in P. putida show low expression strengths and possess only weak controllability. A fundamental understanding of these systems is necessary in order to design robust and predictable biotechnological processes. To fill this gap, we established and characterized a modular T7 RNA polymerase-based system for heterologous protein production in P. putida, using the enhanced Green Fluorescent Protein (eGFP) as an easy-to-quantify reporter protein. We have effectively targeted the limitations associated with the initial genetic setup of the system, such as slow growth and low protein production rates. By replacing the T7 phage-inherent TΦ terminator downstream of the heterologous gene with the synthetic tZ terminator, growth and protein production rates improved drastically, and the T7 RNA polymerase system reached a productivity level comparable to that of an intrinsic RNA polymerase-based system. Furthermore, we were able to show that the system was saturated with T7 RNA polymerase by applying a T7 RNA polymerase ribosome binding site library to tune heterologous protein production. This saturation indicates an essential role for the ribosome binding sites of the T7 RNA polymerase since, in an oversaturated system, cellular resources are lost to the synthesis of unnecessary T7 RNA polymerase. Eventually, we combined the experimental data into a model that can predict the eGFP production rate with respect to the relative strength of the ribosome binding sites upstream of the T7 gene.

Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches.

The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581 T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed.

Patient- and treatment-related risk factors for nausea and emesis during concurrent chemoradiotherapy.

PURPOSE: To evaluate the prevalence of acute nausea and emesis during concurrent chemoradiotherapy (CRT) with emphasis on the influence of patient- and treatment-related risk factors and prophylactic antiemetic medication. PATIENTS AND METHODS: A total of 335 patients treated with different intravenous standard chemoradiotherapy protocols in the inpatient setting were included in this retrospective study. Acute nausea and emesis, scored according to the CTC (version 3.0) criteria, were evaluated during 821 chemotherapy cycles. Side effects were correlated with patient-, tumor-, and treatment-related parameters. RESULTS: Overall, at least one episode of acute nausea occurred in 48% of the patients and at least one episode of vomiting occurred in 25% of patients. The emetogenic level of the applied chemotherapy protocol was the most significant risk factor for developing nausea and emesis (p < 0.0001). The site of irradiation - namely the thorax (p = 0.0110) and head and neck (p = 0.0415) - was also confirmed as a risk factor. Patient-related parameters, e.g., female gender (p = 0.0003), young age (< 40 years; p = 0.0029), weight loss > 5% (p = 0.0004), and the presence of a percutaneous endoscopic gastrostomy (PEG; p = 0.0071), were associated with higher rates of nausea and emesis, while a history of alcohol abuse showed a protective effect (p = 0.0553). In high emetogenic chemotherapy protocols, prophylaxis with 5-HT3 antagonist plus dexamethasone was superior to 5-HT3 antagonist alone (p = 0.0383). CONCLUSION: Future studies should evaluate more effective prophylaxis protocols in CRT in order to reduce the high rates of nausea and emesis.

Trehalose production by Cupriavidus necator from CO2 and hydrogen gas.

In this work, the hydrogen-oxidizing bacterium Cupriavidus necator H16 was engineered for trehalose production from gaseous substrates. First, it could be shown that C. necator is a natural producer of trehalose when stressed with sodium chloride. Bioinformatic investigations revealed a so far unknown mode of trehalose and glycogen metabolism in this organism. Next, it was found that expression of the sugar efflux transporter A (setA) from Escherichia coli lead to a trehalose leaky phenotype of C. necator. Finally, the strain was characterized under autotrophic conditions using a H2/CO2/O2-mixture and other substrates reaching titers of up to 0.47 g L-1 and yields of around 0.1 g g-1. Taken together, this process represents a new way to produce sugars with high areal efficiency. With further metabolic engineering, an application of this technology for the renewable production of trehalose and other sugars, as well as for the synthesis of 13C-labeled sugars seems promising.

Immobilization of PETase enzymes on magnetic iron oxide nanoparticles for the decomposition of microplastic PET.

Polyethylene terephthalate (PET) is responsible for a large amount of environmental contamination with microplastics. Based on its high affinity, the PET degrading enzyme PETase can be immobilized on superparamagnetic iron oxide nanoparticles through a His-tag. The His-tag increases enzyme stability, and allows magnetic separation for recovery. Multiple recycling steps are possible and microplastic particles can be decomposed depending on the PET's crystallinity. The separation or decomposition of PET allows for a sustainable way to remove microplastic from water.

A computed tomography vertebral segmentation dataset with anatomical variations and multi-vendor scanner data.

With the advent of deep learning algorithms, fully automated radiological image analysis is within reach. In spine imaging, several atlas- and shape-based as well as deep learning segmentation algorithms have been proposed, allowing for subsequent automated analysis of morphology and pathology. The first "Large Scale Vertebrae Segmentation Challenge" (VerSe 2019) showed that these perform well on normal anatomy, but fail in variants not frequently present in the training dataset. Building on that experience, we report on the largely increased VerSe 2020 dataset and results from the second iteration of the VerSe challenge (MICCAI 2020, Lima, Peru). VerSe 2020 comprises annotated spine computed tomography (CT) images from 300 subjects with 4142 fully visualized and annotated vertebrae, collected across multiple centres from four different scanner manufacturers, enriched with cases that exhibit anatomical variants such as enumeration abnormalities (n = 77) and transitional vertebrae (n = 161). Metadata includes vertebral labelling information, voxel-level segmentation masks obtained with a human-machine hybrid algorithm and anatomical ratings, to enable the development and benchmarking of robust and accurate segmentation algorithms.

Engineering sucrose metabolism in Pseudomonas putida highlights the importance of porins.

Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf-5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose - making it the fastest growing, plasmid-free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild-type P. putida. This can be seen as a further step towards the production of low-value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future.

A Nitrate-Blind P. putida Strain Boosts PHA Production in a Synthetic Mixed Culture.

One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. In this context, cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals. Based on this, we recently presented a synthetic bacterial co-culture for the production of medium-chain-length polyhydroxyalkanoates (PHAs) from CO2. This co-cultivation system is composed of two partner strains: Synechococcus elongatus cscB which fixes CO2, converts it to sucrose and exports it into the culture supernatant, and a Pseudomonas putida strain that metabolizes this sugar and accumulates PHAs in the cytoplasm. However, these biopolymers are preferably accumulated under conditions of nitrogen limitation, a situation difficult to achieve in a co-culture as the other partner, at best, should not perceive any limitation. In this article, we will present an approach to overcome this dilemma by uncoupling the PHA production from the presence of nitrate in the medium. This is achieved by the construction of a P. putida strain that is no longer able to grow with nitrate as nitrogen source -is thus nitrate blind, and able to grow with sucrose as carbon source. The deletion of the nasT gene encoding the response regulator of the NasS/NasT two-component system resulted in such a strain that has lost the ability use nitrate, but growth with ammonium was not affected. Subsequently, the nasT deletion was implemented in P. putida cscRABY, an efficient sucrose consuming strain. This genetic engineering approach introduced an artificial unilateral nitrogen limitation in the co-cultivation process, and the amount of PHA produced from light and CO2 was 8.8 fold increased to 14.8% of its CDW compared to the nitrate consuming reference strain. This nitrate blind strain, P. putidaΔnasT attTn7:cscRABY, is not only a valuable partner in the co-cultivation but additionally enables the use of other nitrate containing substrates for medium-chain-length PHA production, like for example waste-water.